Cell culture is widely used today for the production of various biologically active products, such as viral vaccines, monoclonal antibodies, non-antibody immunoregulators, polypeptide growth factors, hormones, enzymes, tumor specific antigens, etc. These products are produced by normal, transformed and genetically engineered cells.
For culturing of cells, it is essential to supplement the culture medium with serum, which serves as a universal nutrient for the growth of all cell lines, as well as for the production of most biologically active products. Serum contains hormones, growth factors, carrier proteins, attachment and spreading factors, nutrients, trace elements, etc. Culture medium usually contains up to about 10% of animal serum, such as fetal bovine serum (FBS).
Although widely used, serum has many limitations. It contains high levels of numerous proteins which interfere dramatically with the small quantities of the desired proteins produced by the cells. These serum proteins must be separated from the product during downstream processing which complicates the process and increases the cost. Another limitation is the serum batch-to-batch inconsistencies, resulting in serious regulatory concern about various serum protein contamination in the product.
Recently the advent of BSE (Bovine Spongiform Encephalopathy), a transmissible neurodegenerative disease of cattle with a long latency or incubation period, has further raised regulatory concerns about using animal-derived sera in the production of biologically active products.
A further shortcoming of employing animal sera, such as FBS, is its unsteady supply due to the increase in demand, which in turn causes upwards fluctuations in its price.
There is therefore a great demand for the development of an alternative medium supplement to support cell growth and production of biologically active products.
The advantages and disadvantages of serum-free culture for the manufacture of recombinant biopharmaceuticals from mammalian cells has been thoroughly reviewed (Barnes, 1987; Barnes & Sato 1980; Broad, et al., 1991; Jayme, 1991). The list of the main additives which are used as supplements for serum-free media is summarized by Barnes (1987) and Barnes & Sato (1980).
Unlike serum-supplemented medium, which may be utilized for a broad range of cell types and culture conditions, serum free formulations are generally highly-specific (Barnes, et al. 1984, Sato, et al. 1982, Taub, 1985, Weiss, et al., 1980).
Most commercially available serum-free media contain carrier protein, such as albumin. The presence of carrier protein might be required for protection of the cell viability, but has the afore-mentioned disadvantages for the purification process.
CHO cells have emerged as an appropriate recipient mammalian host to accommodate transfection and expression of a variety of foreign gene products for potential diagnostic and therapeutic applications (Familletti and Fredericks, 1988, Marino, 1989).
A number of commercial serum-free media are available for CHO cell culturing. However, these suffer from multiple disadvantages. Most are suitable for small-scale laboratory applications but become too expensive for large-scale bioreactors. Some are appropriate for cell growth, but perform poorly as production medium. Each of these media might be suitable for the specific system for which it was developed, but cannot usually be used in other systems.
One known serum free culture medium (U.S. Pat. No. 5,063,157) for non-adherent mammalian cells comprises, in addition to the base medium, transferrin, insulin, a peptone, a beta-D-xylopyranose derivative, selenite and a biological polyamine. The only disclosure in the above patent relating to production concerns culturing of specific mouse hybridoma cells in a medium, which, in addition to the base medium, contains six ingredients. Production in any other mammalian cell of a biologically active product, other than an FSH antibody, is not taught.
Another serum free cell growth medium for mammalian cells is disclosed in U.S. Pat. No. 4,443,546. This growth medium, in addition to the basic medium, contains seven ingredients.
European patent specification No. 481,791 discloses a culture medium for CHO cells comprising water, an osmolality regulator, a buffer, an energy source, amino acids, an iron source, a growth factor and other optional components. The two media exemplified contain 19 and 17 components, respectively.
The major objectives in development of a serum-free medium for large-scale production are: a serum-free, protein-free (or low protein), defined medium with minimal additives resulting in a lower cost, effective medium, which does not contain ingredients that are likely to complicate the culturing/production/purification process steps.
If proteins are nevertheless present in the medium, it is preferred that they are obtained by recombinant means and not isolated directly from an animal source.
Although the list of known potential additives to serum free media is very long, the correct combination has not yet been found in many cases. Indeed, there are only about 40 commercial serum-free media on the market, despite the fact that the need therefor has been known for more than a decade.